X-ray diffraction and other scattering techniques (e.g. neutron diffraction) are powerful characterization tools for providing 3-dimensional structural information on the scale of atoms for crystalline materials. Such coherent scattering methods fail for many soft, disordered materials, and optical methods for structural determination, such as laser scanning confocal fluorescence microscopy, are used to determine 3-dimensional structure at length scales >1 μm. However, at smaller length scales, optical methods are limited in resolution by the wavelength of light used for observation (approximately 500 nm for visible light).
Optical microscopy methods exceeding the diffraction limit of light by relying on the fact the central position of a single fluorescent molecule (fluorophore) can be determined to a much higher accuracy (presently, having precision ˜2 nm) than the width of its diffraction-limited image have been investigated, and will likely attain wide-spread use in the exploration of the finer details of cellular structure. In addition to biological structures, these single-molecule localization microscopy techniques hold promise for nano-scale imaging of many materials. In contrast to electron microscopy, single molecule localization microscopy is applicable to materials that have little or no contrast in electron density and for materials immersed in a wet or aqueous environment. Moreover, in contrast to surface-specific scanning probe techniques, such as near-field scanning optical microscopy, single-molecule localization microscopy has the potential for enabling full 3-D imaging of materials at near nanometer resolution.
As an example, while quantum dots have a diameter of 10 nm, diffraction blurs the image of a single quantum dot into a circular “blob” a few hundred nanometers across. However, one can determine the central position of the blob to a much greater accuracy than the width of the blob, with 2 nm resolution obtainable under certain favorable circumstances. Using the fact that a single fluorophore can be located in 2 dimensions with this high accuracy, one can generate a pseudo image using known calculation techniques, such that each point in this new image has a width given by the uncertainty in the determining the central position of the original diffraction limited spot. When these localization methods are combined with a method for selectively activating a small number of fluorophores for imaging, high resolution images (in two spatial dimensions) that reflect the underlying molecular density can be obtained.
Photo-activated or photo-switchable fluorophores and single-molecule detection are utilized for such high-resolution imaging methods, as molecules must be individually imaged such that their diffraction limited spots do not overlap in the image plane. For structures densely labeled with a fluorescent species, imaging individual molecules is accomplished by using a photo-activatible or photo-switchable fluorescent reporter, such as photo-activatible green fluorescent protein, PA-GFP. PA-GFP is normally in a dark (non-fluorescent) state, but can be activated into a fluorescent state upon excitation with ultra-violet radiation. In photoactivation localization microscopy, (PALM), these photoswitchable proteins are fused to a structural protein of interest. A weak UV light pulse is then applied to activate only a few molecules. These molecules are imaged, their central locations calculated, and the activated molecules are subsequently photo-bleached. Another UV light pulse is then applied to activate a few more molecules, beginning another imaging cycle. This activation, imaging, and bleaching process is repeated many times. The results from each imaging cycle are combined to form a composite pseudo-image from the entire data set. A review of the PALM procedure may be found in “Imaging Intracellular Fluorescent Proteins At Nanometer Resolution” by Eric Betzig et al. in Science 313 (15 Sep. 2006), 1642-1645, wherein photoactivatable green fluorescent protein (PA-GFP) was employed. The single-molecule localization microcopy method of Betzig et al. was limited to 2D slices.
In addition to activation of PA-GFP with ultraviolet radiation, one can also activate this molecule via two-photon excitation methods. In this activation method, two photons of near-infrared wavelengths (˜800 nm) are used to convert the chromophore from a non-fluorescent to a fluorescent state. The two-photon activation of PA-GFP was recently demonstrated in “Two-Photon Activation And Excitation Properties Of PA-GFP In The 720-920-nm Region” by Marc Schneider et al. in Biophysical J. 89 (August 2005) 1346-1352. Further, in “Two-Photon Laser Scanning Fluorescence Microscopy” by Winfried Denk et al. in Science 248 (6 Apr. 1990) 73-76, molecular excitation by the simultaneous absorption of two photons in laser scanning fluorescence microscopy is described.
Another useful photoactivatable molecule is caged fluorescein which has large nitrophenyl rings that cleave off the molecule upon excitation with UV light, increasing the fluorescence quantum yield of the fluorophore ˜500 fold. See, e.g., T. J. Mitchison in “Polewards Microtubule Flux In The Mitotic Spindle Evidence From Photoactivation Of Fluorescence,” J. Cell Biology 109 (1989) 637-652. Caged fluorescein has a bright/dark ratio of 500:1, whereas PA-GFP has a ratio closer to 100:1. This superior bright/dark ratio for caged fluorescein is one reason this molecule is a promising contrast reagent for high resolution localization microscopy. Other reasons include the fact that caged fluorescein is commercially available (with or without an amine-reactive linker), and there is a large difference in fluorescence lifetimes between the caged (dark) and uncaged (bright) forms of this molecule. The fluorescence lifetime of the caged/quenched dye is ˜210 ps, whereas the activated fluorophore has a fluorescence lifetime of 3.4 ns. This difference in fluorescence lifetime means time-gating can dramatically reduce the contribution of un-activated molecules to the background; for example, if one applies a >2-ns time-gate to the data, the fraction of photons that pass through the time-gate for the activated molecule is ˜0.55 (e−2/3.4), whereas the fraction of the photons that pass through the time-gate for the un-activated (quenched) fluorophores also present in the field of view is almost four orders of magnitude less, ˜0.00007 (e−2/0.21). This reduction in background is essential for high-resolution imaging of samples densely labeled with fluorescent reporters, as will be encountered in any sample labeled with the appropriate contrast agent throughout a volume of hundreds of microns.
The optical resolution is therefore not limited the wavelength of light used for observation, but rather by the accuracy an individual fluorescent molecule can be located. This localization accuracy is typically around 20 nm, and depends upon the total number of photons emitted by the molecule prior to photobleaching and the background noise level the molecule is imaged upon. It can be shown that the localization accuracy is strongly dependent upon the background—molecules where only a few photons are detected can still be located to sub-20 nm precision, if done so in a low background environment. Therefore, reducing the background light is important for increasing localization accuracy for a fixed image integration period.
The primary background in these measurements comes from un-activated molecules in the field of view. In particular, an un-activated PA-GFP is not completely dark. As stated hereinabove, the ratio of the fluorescence from an activated PA-GFP to an un-activated PA-GFP is roughly 100:1. The construction of a high-resolution PALM image requires a large density of activatable molecules. For example, assuming that the single molecule stipple points in a final PALM image are ˜50 nm apart, then, prior to any activation, a small 0.5 by 0.5 μm field of view contains ˜100 photoactivatable fluorophores, which provides a weak fluorescence background. If a single fluorophore in this window is activated on the first PALM cycle, and 250 photons from the activated molecule are measured before it bleaches, the detected photons are measured with a background of approximately an additional 250 photons due to the 100 weakly emitting un-activated molecules also present in the small fitting window. The localization accuracy for this molecule would be quite poor (˜320 nm), and the molecule would not be used in the final image reconstruction. Rather, a composite PALM image is formed from “hand-picked” molecules that lie in the tail of two distributions: (a) those that emit a large number of photons prior to bleaching; and (b) the last molecules activated which are imaged in a nearly background-free environment, as all other fluorescent molecules in the field of view have, by this time, photo-bleached. This “cherry-picking” for finding favorable molecules limits the speed of image acquisition: as originally developed, each PALM image required ˜1 day to acquire, although use of superior fluorescent proteins has decreased this time to approximately 2 minutes per image.
In “Time-Gated Biological Imaging By Use Of Colloidal Quantum Dots” by M. Dahan et al., Optics Letters 26 (1 Jun. 2001) 825-827, the long fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. The inorganic quantum dots emit light slowly enough that most of the autofluorescence background is over by the time emission occurs, but fast enough to maintain a high photon turnover rate.
Advances in this field have extended single-molecule localization methods to three dimensions. In “Tracking of Single Fluorescent Particles In Three Dimensions: Use Of Cylindrical Optics To Encode Particle Position” by H. Pin Kao and A. S. Verkman in Biophysical J. 67 (September 1994) 1291-1300, and in “Three-Dimensional Super-Resolution Imaging By Stochastic Optical Reconstruction Microscopy” by Bo Huang et al. 319 Science (8 Feb. 2008) 810-813, 3D stochastic optical reconstruction microscopy (STORM) has been demonstrated using astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy using optical astigmatism or ellipticity generated by introducing a cylindrical lens into the imaging path. Bo Huang et al. describe obtaining ˜30 nm resolution in 3 spatial dimensions, limited to sparsely labeled cellular structures over a z-depth of less than 1 μm.